The acute effects of repetitive transcranial magnetic stimulation on laminar diffusion anisotropy of neocortical gray matter

Abstract Repetitive transcranial magnetic stimulation (rTMS) is increasingly used to treat neuropsychiatric disorders. Inhibitory and excitatory regimens have been both adopted but the exact mechanism of action remains unclear, and investigating their differential effects on laminar diffusion profiles of neocortex may add important evidence. Twenty healthy participants were randomly assigned to receive a low‐frequency/inhibitory or high‐frequency/excitatory rTMS targeting the left dorsolateral prefrontal cortex (DLPFC). With the brand‐new submillimeter diffusion tensor imaging of whole brain and specialized surface‐based laminar analysis, fractional anisotropy (FA) and mean diffusion (MD) profiles of cortical layers at different cortical depths were characterized before/after rTMS. Inhibitory and excitatory rTMS both showed impacts on diffusion metrics of somatosensory, limbic, and sensory regions, but different patterns of changes were observed—increased FA with inhibitory rTMS, whereas decreased FA with excitatory rTMS. More importantly, laminar analysis indicated laminar specificity of changes in somatosensory regions during different rTMS patterns—inhibitory rTMS affected the superficial layers contralateral to the DLPFC, while excitatory rTMS led to changes in the intermediate/deep layers bilateral to the DLPFC. These findings provide novel insights into acute neurobiological effects on diffusion profiles of rTMS that may add critical evidence relevant to different protocols of rTMS on neocortex.

on diffusion profiles of rTMS that may add critical evidence relevant to different protocols of rTMS on neocortex.

K E Y W O R D S
diffusion tensor imaging, dorsolateral prefrontal cortex, fractional anisotropy, magnetic resonance imaging, mean diffusion, repetitive transcranial magnetic stimulation INTRODUCTION As a noninvasive brain stimulation technique, transcranial magnetic stimulation (TMS) has emerged as an alternative interventional approach for patients with neuropsychiatric and neurological disorders who have not achieved optimal outcomes after first-line pharmacological treatments. [1][2][3] Repetitive TMS (rTMS) is known to exert therapeutic effects by inducing an electromagnetic field in the brain that results in a strong and rapidly fluctuating electrical current within the neocortex, which in turn depolarizes neurons and modulates cortical excitability at the site of stimulation. 1,4,5 However, the exact neurophysiological mechanism of rTMS action remains to be fully delineated. Though it has been suggested that modulating the metaplasticity and inducing the synaptic plasticity might be one of the ways that rTMS exerts the effects, 6 uncertainty about the full range of effects of TMS on the brain has led to some conflicting evidence on systematic application of rTMS in some psychiatric and cognitive disorders 7,8 and slowed a wider clinical use of this novel and effective therapeutic strategy. 9,10 In the past two decades, neuroimaging studies have provided insights into how TMS affects brain in anatomy, function, and chemistry. For example, magnetic resonance spectroscopy studies have demonstrated changes in γ-aminobutyric acid concentrations in relation to TMS-based changes in brain electrophysiology, 11,12 and positron emission tomography studies of regional cerebral blood flow/glucose metabolic rate have shown changes after long stimulation. 13 Functional magnetic resonance imaging (fMRI) has also been used to document the acute impact of TMS on brain networks, 14 which has shown that rTMS-induced functional changes tend to spread distally across and within networks. 15,16 Recently, a concurrent interleaved TMS/fMRI study was conducted with state-of-the-science purpose-designed MRI head coils to delineate networks and downstream regions activated by DLPFC-TMS, and it was found that regions of increased acute signal activation during TMS resemble a resting-state brain network. 17 These studies provided important progress highlighting the importance of wider network-level consequences of TMS after site stimula-tion. However, the neurobiological effects of rTMS can be diverse depending on the TMS paradigms 18,19 and differential effects of low-frequency inhibition and highfrequency excitation across brain networks are not well characterized, especially how brain changes that occur distal to the site of TMS administration, and the laminar distribution of the effects, remain to be fully understood.
Dual-site TMS studies have demonstrated conditioning effects in cortico-cortical pathways when stimulating two regions, 13 and changes in such pathways may be related to modifications of microstructural properties including neurite density or myelination of fibers in gray matter, 20,21 or osmotic changes in axons due to altered cell firing rates or in extracellular water content related to perivascular changes, which can be measured with diffusion MRI. 22 Therefore, investigating structural changes across brain gray matter after acute TMS that can be evaluated by diffusion tensor imaging (DTI), which assesses the directional diffusion of water along axon tracts might provide novel information relevant to the widely distributed effects of rTMS on the neocortex. 23 Especially, in clinical psychiatry, the dorsolateral prefrontal cortex (DLPFC) is the most common target of TMS. Therefore, characterizing how stimulation of DLPFC causes network-wide changes might provide critical information relevant to the full mechanistic understanding of its therapeutic efficacy.
Recent progress in image acquisition, such as the multiplexed sensitivity-encoding (MUSE) technique, 24 adopts a multishot scan strategy to improve image resolution and reduce distortion and blurring artifacts simultaneously. This technique has made it possible to collect high-resolution whole-brain DTI data within half an hour to evaluate coherent patterns of diffusion anisotropy in the gray matter at the submillimeter level. In combination with high-resolution neuroimaging, surfacebased analysis has helped to identify the architectonic domains of diffusion orientation in the human cortex. 21 These advances provide a useful tool for characterizing TMS-related brain changes at the laminar level in a more manageable scan duration period, which allows for investigating rTMS effects across superficial, intermediate, and deep cell layers, which have different input, output, and cytoarchitectonic features, as are studies examining changes across infragranular, granular, and supragranular layers. 25 When comparing "excitatory" and "inhibitory" stimulation, to the degree that differences in brain effects of these two rTMS approaches are due to a change in cell firing rates, then inverse effects of excitatory and inhibitory rTMS would be predicted, and then demonstrating such opposite effects would add confidence to data interpretation regarding changes in MRI features being related to changes in neuronal excitation during the TMS.
With these considerations in mind, a cohort of healthy participants was recruited and randomly assigned to either receiving excitatory or inhibitory rTMS, with interleaved MUSE-DTI data collection to obtain whole-brain diffusion data at high resolution (0.9 mm isotropic acquisition). Then, a surface-based laminar analysis was performed to enable sampling of DTI signals within cortical layers as in previous work 21 in order to quantify and compare the acute changes of diffusion anisotropy architecture related to rTMS excitation and inhibition. The "acute" here refers to the immediate MUSE-DTI data collection after the rTMS stimulation which could offer a timely readout of "acute" rTMS effect in contrast to clinically observed effects that usually occur weeks after rTMS. 26 We found that rTMSinduced changes in diffusion anisotropy of neocortical gray matter varied in their depth across specific regions and were in opposite directions for excitatory and inhibitory rTMS. This characterization of the spatial characteristics of TMS-induced brain changes at the laminar and regional level in water diffusion profiles may help clarify the differential effects of rTMS paradigms on the neocortex that future applications might refer to for specific treatment purpose.

Demographic characteristics
In order to measure the acute changes of diffusion anisotropy architecture related to different rTMS patterns, 20 healthy participants were randomly assigned to receiving a single session of low-frequency inhibitory rTMS (subgroup 1, n = 9, seven females, age: 23.56 ± 2.41 years, education: 16.89 ± 1.54 years) or high-frequency excitatory rTMS (subgroup 2, n = 11, nine females, age: 25.82 ± 2.56 years, education: 19.18 ± 2.04 years), both targeting left DLPFC. A within-subject cross-over design was not used to avoid the potential effects of prior stimulation on response to the second stimulation. Participants were scanned immediately before and after the rTMS session, and laminar analysis was conducted with the collected MRI data. The flowchart of study procedures is presented in Figure 1. There were no significant differences in age or sex distribution between groups, while the latter had a significantly higher educational level than the former (Table 1).

Effects of rTMS inhibition on diffusion metrics in laminar cortex
To characterize the acute effects of inhibitory stimulation on diffused properties of laminar cortex, a paired-sample t-test was performed to obtain the laminar fractional anisotropy (FA) and mean diffusivity (MD) changes in cortical gray matter before and after the inhibitory rTMS.  (Table  S1). In contrast, laminar profiles in multiple frontal lobe regions showed increased MD values, including bilateral DLPFC (right BA 9 and left BA 46), bilateral anterior prefrontal cortex (bilateral BA 10), and left lateral and medial premotor cortex (BA 6) and regions of left BA 8. Most of these changes were observed at superficial to middle layers within gray matter (Table S2).
These results indicated that cortical regions with significant changes in FA and MD were mainly located in somatosensory, limbic, and sensory regions after inhibitory rTMS. Specifically, cortical regions only showed increased FA after inhibitory rTMS, and the conditioning F I G U R E 1 Flowchart of data acquisition and laminar analysis. During the data acquisition, participants were scanned with high-resolution MRI at baseline (Step 1) and randomly divided into two subgroups (Step 2). Participants in subgroup 1 and subgroup 2 each received inhibitory or excitatory rTMS targeting the left DLPFC after the baseline MR scan (Step 3) and were scanned again immediately after the rTMS session (Step 4). After data collection, laminar analysis was then performed. The preprocessed high-resolution T1-weighted images were used to generate the surface of pial and white matter (Step 5, the reconstructed pial surface is marked with yellow line, while the white matter surface is white line). The layer segmentation based on the surfaces above was conducted and the reconstruction of the segmentation was superimposed as red ribbon (Step 6). Equal-distance method was adopted and 11 layers based on 10 intermediate surfaces were evenly generated and superimposed as ribbons with different colors (Step 7). The DTI data were registered with the laminar information built with high-resolution T1-weighted images (Step 8, each line overlaid on the DTI axial slice represented a specific layer and 5 layers were shown here as sketch map). MRI-magnetic resonance imaging, DTI-diffusion tensor imaging, rTMS-repetitive transcranial magnetic stimulation, DLPFC-dorsolateral prefrontal cortex. effects induced by inhibitory rTMS were observed in the superficial layers of somatosensory regions contralateral to the DLPFC.

Effects of rTMS excitation on diffusion metrics in laminar cortex
The same analysis was also adopted to characterize the acute effects of excitatory stimulation. After excitatory rTMS, participants showed decreased FA values in widespread regions, including bilateral primary somatosensory cortex ( showed increased MD values at superficial to middle layers within gray matter (Table S4). These results indicated that cortical regions with significant changes in FA and MD were also located in somatosensory, limbic, and sensory regions after excitatory rTMS. Nevertheless, cortical regions only showed decreased FA after excitatory rTMS, and the conditioning effects induced by excitatory rTMS led to changes in the intermediate/deep layers of somatosensory regions in both hemispheres.

Difference in diffusion metrics after rTMS excitation and inhibition
To compare the effects of excitation and inhibition stimulation, differences (△values = values after simulation − val-ues at baseline) in FA and MD values after rTMS stimulation were compared across the two study groups using two-sample t-tests.  Tables S5 and S6.
These findings showed that participants after rTMS inhibition had larger increases in FA and larger alternations in MD in several regions than participants who had excitatory rTMS by directly comparing the changes in FA and MD values before and after each pattern of rTMS.

DISCUSSION
In this study, to contrast the acute effects of rTMS excitation and inhibition on neocortical gray matter, we performed a laminar analysis with high-resolution whole-brain diffusion data. Several significant findings were demonstrated. First, brain regions with diffusion anisotropy changes were widely distributed in regions distal from the stimulation site, mainly in somatosensory regions, limbic regions, and some sensory regions, after both TMS excitation and inhibition. Second, regions with changes in diffusion measures after inhibitory rTMS all showed increased FA values, and most of these effects were contralateral to the stimulating site. In contrast, after excitatory rTMS, regions with altered diffusion measures all showed decreased FA values and the affected regions were most often ipsilateral to the stimulation site (rTMS excitation also decreased FA values of ipsilateral frontal regions). Third, the early post-TMS cortico-cortical pathways underlying conditioning effects appear to involve the superficial layers of somatosensory regions contralateral to the stimulating site of inhibition, and the intermediate/deep layers of these regions bilaterally near the stimulating site of excitation. Effects were also seen across all layers in some sensory regions, most notably in the middle layers within gray matter. The first important finding of our study is that both inhibitory and excitatory rTMS consistently and mainly stimulate diffusion profile changes in somatosensory regions, limbic regions, and some sensory regions. This suggests that the changes in diffusion anisotropy induced by rTMS are widespread, well beyond the site of stimulation, which highlights how focal rTMS can have broadranging impact on brain regions/networks involved in emotion and cognitive processing. 27 The activated regions via our rTMS paradigms and diffusion measures were quite similar to those recently measured with functional MRI, 17 in which brain regions in DLPFC, inferior parietal lobe, and anterior cingulate cortex all have been observed with increased acute signal activation during TMS. 17 More importantly, in a study that investigated treat-ment effects of rTMS with DLPFC targeting in depression, these regions have also been observed with alterations after a seed-based connectivity analysis. 28 Notably, somatosensory regions are related to proprioception, 29 while limbic regions including cingulate cortex usually are in charge of receiving inputs or external stimulations from other brain regions and involved in complicated and interconnected cognitive and emotional processing. 30 While the TMS has been suggested to act through a top-down mechanism, 27 it could be inferred that some specific neural circuit is evoked after DLPFC stimulation, in which somatosensory regions percept the stimulations whereas the limbic regions TA B L E 3 The alternations of fractional anisotropy values after rTMS excitation. process them. Further studies are warranted for the precise characterization of the abovementioned physiologic processes in order to understand their potential contribution to treating cognitive and emotional processing impairments.

Mean (SD)
The second important finding is that the specific changes of FA after TMS excitation and inhibition were in opposite directions. Several mechanisms might underpin these changes. First, FA values in part reflect myelination of axons, 31 which in the cortex are aligned perpendicular to the cortical surface. [32][33][34] Collateral branches of axons and dendrites of neurons branch in less organized ways, but the primary large axons are organized directionally to provide a directional diffusion signal. 35 In previous studies of multiple sclerosis, the loss of these axons and their myelination leads to a decrease of FA values in the neocortex, while FA values in some regions can be increased due to the concomitant loss of axonal branching. 34 While these findings highlight the biological significance of diffusion imaging in gray matter, such changes seem unlikely to account for the observed diffusion changes after acute rTMS. Aside from rapid changes in myelination, regional changes in neuronal membrane permeability or water volume content in pyramidal cell axons following marked increases (excitatory rTMS) and decreases (inhibitory rTMS) in their cell firing rates during TMS might also contribute to the acute regional diffusion changes. High-frequency and low-frequency TMS treatment depolarize different kinds of neurons 18 leading to changes in neurophysiology outside the normal range of cell firing rates, which could cause acute neuronal swelling via mechanisms that would differentially impact aggregate FA measurements. [36][37][38] Another mechanism that might contribute to the difference of FA changes after excitatory and inhibitory rTMS is the differential neurotransmitter concentration across cortical layers. A recent examination of the glutamate signal changes in response to theta-burst stimulation indicated a pattern of decreases in the ipsilateral hemisphere. 11 This might underpin our observed changes that after rTMS excitation, the FA values of ipsilateral regions were more likely to change while after rTMS inhibition, the FA values of contralateral were more likely to change. The distinct effects of rTMS were attributed to those fibers within the cortical region stimulated which projected ortho-or antidromically to distant structures. 39,40 However, more work is need to clarify the fundamental neurobiological cause of such effects, and their duration.
The third important finding is that we observed increased FA in superficial layers of somatosensory regions after inhibition but decreased FA in deeper layers after excitation, while the limbic and sensory regions showed no laminar specificity of FA changes. Such laminar differences suggest that different cortical layers in somatosensory regions react differently to rTMS frequency, while the cause of such effects and potential clinical implications remains to be determined. Notably, though the segmented lamina does not correspond directly to cortical layers which are cytoarchitecturally defined and vary their spacing across neocortex, our analysis does suggest that superficial and deep layers in these regions respond quite differently to excitatory and inhibitory rTMS.
In addition to diffusion metric changes in the brain regions as noted above, there were also FA or MD alterations in frontal cortex, mainly in intermediate and superficial layers. The FA values were found to decrease in several left frontal regions after rTMS excitation, and increased MD was demonstrated mainly in left (ipsilateral) frontal regions after rTMS inhibition. Such findings suggested that after stimulating left DLPFC, other frontal regions can also be simulated as they are interconnected part of intrinsic functional brain networks of frontal cortex. 15 The MD changes are noteworthy, as MD measures are sensitive to cellularity and edema, 41 so that MD in frontal regions after inhibition might manifest as a result of subtle brain edema. While our findings demonstrate that focal rTMS can have wide ranging effects on the anatomy of neocortical gray matter, putatively by changing cell firing rates to extraphysiological levels, whether these acute changes represent clinically relevant adverse effects and whether they persist beyond the immediate post rTMS period remain to be determined.
Some limitations are noteworthy regarding the current study. First, we did not include a sham control group to rule out the possibility that the results might be confounded by nonspecific effects of rTMS. Our observations of opposite changes after excitatory and inhibitory rTMS reduce concerns about such artifacts to a degree. Second, we only examined the rTMS effects after single stimulation in left DLPFC. While this is a common stimulation site in clinical practice, effects following rTMS to other brain regions remain to be determined. Third, in our laminar analysis, the layers do vary as a percent of depth in neocortex, they do not directly correspond with histologically defined cortical layers I-VI. Examination of effects in cytoarchitecturally defined layers and clarification of causes of diffusion changes might best be obtained from future preclinical research with animal models. Fourth, the sample size of current analysis is not large and the sex distributions were not balanced (there were only two male participants in each group). Therefore, our findings should be considered with caution. Replication of our findings in larger studies is needed in the future. Fifth, though we characterized the rTMS in each participant group, the difference in education level might also confound the findings, which should be taken into consideration in future work especially when two participant groups are directly compared. Finally, studies from the clinical perspective need to confirm that our findings do not reflect clinically meaningful adverse effects on motor, sensory, affective or cognitive function, and the persistence of the findings needs to be examined with longer term follow-up studies.

Participants
This study was approved by the Research Ethics Committee of West China Hospital and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants. In this study, twenty healthy participants were recruited through poster advertisements from the nearby Chengdu community. All participants were right-handed and of Chinese Han ancestry. The age range for included participants was 20−31 years. Participants were excluded if they had: (1) contraindications to MRI scanning (e.g., claustrophobia or braces), (2) a history of substance abuse or dependence, (3) a positive pregnancy test, (4) a history of major systemic disease, known psychiatric/neurological illness, or an episode of loss of consciousness >10 min, and (5) a history of serious mental illness in their first-degree relatives.
Notably, a large cohort of healthy participants for rTMS recording had been established elsewhere before. 42

Repetitive TMS
Repetitive TMS was delivered using a M-100 Ultimate magnetic stimulator (Shenzhen Yingchi Technology Co. Ltd) and a 70 mm figure 8 coil that stimulated a 2 cm 2 area that extended 2 cm into the cortex. 43 The coil was held tangentially to the scalp with the handle pointing back and away from the midline at 45 • . Brodmann Area 9 (BA 9; MNI coordinates: x = −23.1, y = 37.4, z = 43.0) was used as the cortical landmark of left DLPFC. 44 The neuro-navigation system (QuickVision; Shenzhen Yingchi Technology Co. Ltd) was used for precise localization, 45 with coordinates provided by incorporating pre-stimulation high-resolution brain anatomical scans into the navigator. The resting motor threshold (RMT) was measured before the subsequent rTMS stimulation by stimulating the M1 region of the left cerebral hemisphere in a single stimulation mode and determined by involuntary contraction of the fingers to provide the reference baseline. 46,47 High-frequency rTMS stimulation (≥5 HZ) has been associated predominantly with excitatory effects while low-frequency rTMS (≤5 HZ) stimulation has predominantly inhibitory effects. 48,49 In this study, inhibitory rTMS was applied at 1 Hz with an intensity of 100% of RMT, and with 120 trains of 5 s duration with 5 s between trains. Excitatory rTMS was applied at 15 Hz with an intensity of 80% of RMT, with 100 trains of 1 s duration and 11 s between trains.

Data acquisition
All MRI data were collected on a 3.0T MRI scanner (Signa Premier; GE Healthcare) with a 48-channel head coil. A MUSE technique was used to acquire submillimeter whole-brain isotropic DTI data with parameters as follows During the MRI scan, earplugs and headphones were provided to block background noise, and foam padding around the head minimized head motion. Obtained brain images including both T1WI and DTI were visually inspected by two experienced neuroradiologists, and no scan artifacts or gross brain abnormalities were observed in any participant.

4.4
Data processing and laminar depth analysis DTI data preprocessing, including denoising 51 and eddy current correction, 52 was performed with MRtrix3 (https://www.mrtrix.org/) and FSL (https://fsl.fmrib.ox.ac.uk/fsl/). The preprocessed DTI images were used to generate FA maps with MRtrix3. 53 The DTI images were registered to T1-MPRAGE scans using epi_reg provided in FSL, which incorporates white matter boundary information in the registration process. 54,55 T1-MPRAGE images were preprocessed with FreeSurfer (version 7.1.0, http://surfer.nmr.mgh.harvard.edu/) to generate pial surface and white matter surface maps, which were transformed into volume space with AFNI and SUMA (https://afni.nimh.nih.gov/). The pial surface was defined as the boundary between cortical gray matter and cerebrospinal fluid (CSF). The white matter surface represented the boundary between white matter and cortical gray matter. Finally, the layer segmentation based on pial surface and white matter surface was conducted with LAYNII (https://layerfmri.com/2020/05/ 29/laynii-setup/#more-2378). An equal-distance method was adopted, with 10 intermediate surfaces evenly spaced throughout the cortical depth being identified, 56 as done previously. 21 Finally, 11 layers were obtained, with the while matter surface being referred to as the 1st surface, the next closest surface as the 2nd surface and so on to the pial surface, the most superficial gray matter ( Figure S1). Since cytoarchitectural layers cannot be defined in MRI images, so patterns of effects across defined layers could be informative about supragranular, granular, and infragranual layer of cortex. The predefined Brodmann template that defined 82 cortical regions 57 was adopted in this study. Diffusion anisotropy profiles including FA and MD metrics were analyzed by calculating the average measures of the FA and MD metrics across voxels in each layer of each Brodmann region. This laminar subdivision segments the cortex that is ∼3 mm thick into depth regions ("layers," each layer has about 0.3 mm thickness) that are approximately three times thinner than our 0.9 mm voxels. The first and last layers in the segmentation were discarded because of the potential confounding effects of white matter or CSF nearby.

Statistical analyses
To characterize the acute effects of rTMS on microstructural properties of cortical gray matter within each of the two study groups considered separately, we tested for laminar FA and MD changes in neocortex after excitatory and inhibitory stimulations using paired-sample t-tests. A p value (two-tailed) less than 0.05 was considered to be statistically significant. In addition, p values of each brain region were corrected with false discovery rate (FDR) by using the code "FDR_p = mafdr(p,'BHFDR', true)" in Matlab to correct the effects of multiple layers. The code refers to the Benjamini-Hochberg FDR method, 58 which ranks the p values and adjusts them based on their ranking and controls the FDR at a specified level. To contrast the effects of excitation and inhibition stimulation, changes in FA and MD values after TMS stimulation were compared across the two study groups using two-sample t-tests.

CONCLUSIONS
By characterizing the laminar profiles of cortical diffusion anisotropy induced by rTMS, a rapid change in water diffusion in neocortex especially within brain circuits that are involved in sensory systems, cognition, and emotion processing after either excitatory and inhibitory rTMS. These effects varied across regions in their depth from the cortical surface, and were in opposite directions for excitatory and inhibitory rTMS. These findings provide novel insights into the acute neurobiological effects of different protocols of rTMS administered over prefrontal cortex.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
Drs. Wenjing Zhang and John A. Sweeney consult to VeraSci. Drs. Huilou Liang and Miaoqi Zhang are employees of GE Healthcare, MR Research, Beijing, China. The company has no role in designing and performing the study or analyzing and interpreting the data. The remaining authors have no competing interests or financial support to disclose.

D ATA AVA I L A B I L I T Y S TAT E M E N T
West China Hospital of Sichuan University has an institutional commitment to data sharing. To get access to the data and comply with the terms of our research ethics committee approval, an application to the corresponding author will be required, specifying the geographical extent of sharing.

E T H I C S S TAT E M E N T
All procedures involving human participants were approved by the Research Ethics Committee of West China Hospital of Sichuan University (reference NO. 1016) and performed in accordance with the Declaration of Helsinki. All participants provided written informed consent before study participation.